The invention relates to the area of enzyme regulation More particularly, the invention relates to the regulation of human pyroglutamyl peptidase I.
The brain enzyme pyroglutamyl peptidase catalyzes the proteolysis of thyrotropin releasing hormone (TRH) to form histidyl-proline diketopiperazine, also known as cyclo (His-Pro) or CHP. U.S. Pat. No. 6,090,780. Because of the role of this enzyme in hormone metabolism, there is a need in the art to identify related enzymes which can be regulated to provide therapeutic effects.
It is an object of the invention to provide reagents and methods of regulating a human pyroglutamyl peptidase I This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a method of screening for agents which can regulate the activity of a human pyroglutamyl peptidase I. A test compound is contacted with a polypeptide comprising an amino acid sequence which is at least about 29% a identical to the amino acid sequence shown in SEQ ID NO:4. Binding of the test compound to the polypeptide is detected. A test compound which binds to the polypeptide is thereby identified as a potential therapeutic agent for regulating activity of the human pyroglutamyl peptidase I.
Another embodiment of the invention is a method of screening for agents which regulate an activity of a human pyroglutamyl peptidase I. A test compound is contacted with a polypeptide comprising an amino acid sequence which is at least about 29% identical to the amino acid sequence shown in SEQ ID NO:4. A pyroglutamyl peptidase I activity of the polypeptide is detected A test compound which decreases the pyroglutamyl peptidase I activity is thereby identified as a potential therapeutic agent for decreasing the activity of the human pyroglutamyl peptidase I. A test compound which increases the pyroglutamyl peptidase I activity of the polypeptide is thereby identified as a potential therapeutic agent for increasing the activity of the human pyroglutamyl peptidase I.
Yet another embodiment of the invention is a method of screening for agents which regulate an activity of a human pyroglutamyl peptidase I. A test compound is contacted with a product encoded by a polynucleotide which comprises a nucleotide sequence which is at least 50% identical to the nucleotide sequence shown in SEQ ID NO:3. Binding of the test compound to the product is detected A test compound which binds to the product is thereby identified as a potential therapeutic agent for regulating the activity of the human pyroglutamyl peptidase I.
Even another embodiment of the invention is a method of reducing activity of a human pyroglutamyl peptidase I. A cell is contacted with a reagent which specifically binds to a product encoded by a polynucleotide comprising a nucleotide sequence which is at least 50% identical to the nucleotide sequence shown in SEQ ID NO:3. The activity of the human pyroglutamyl peptidase I is thereby reduced
Another embodiment of the invention is a pharmaceutical composition comprising a reagent which specifically binds to a product encoded by a polynucleotide comprising a nucleotide sequence which is at least 50% identical to the nucleotide sequence shown in SEQ ID NO:3 and a pharmaceutically acceptable carrier.
Another embodiment of the invention is a pharmaceutical composition comprising an expression construct encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:4 and a pharmaceutically acceptable carrier.
Yet another embodiment of the invention is an isolated and purified polynucleotide consisting essentially of the nucleotide sequence shown in SEQ ID NO:3.
Still another embodiment of the invention is an isolated and purified polypeptide consisting essentially of the amino acid sequence shown in SEQ ID NO:4.
Even another embodiment of the invention is a preparation of antibodies which specifically binds to a polypeptide consisting essentially of the amino acid sequence shown in SEQ ID NO:4.
A further embodiment of the invention is a method of preparing a polypeptide consisting essentially of the amino acid sequence shown in SEQ ID NO:4. A host cell comprising an expression construct encoding the polypeptide is cultured under conditions whereby the polypeptide is expressed. The polypeptide is isolated.
The invention thus provides a human pyroglutamyl peptidase I which can be used to identify test compounds which may act, for example, as activators or inhibitors at the enzyme""s active site. Human pyroglutamyl peptidase I and fragments thereof also are useful in raising specific antibodies which can block the enzyme and effectively reduce its activity.